TY - JOUR
T1 - Mutational analysis of Kaposica reveals that bridging of MG2 and CUB domains of target protein is crucial for the cofactor activity of RCA proteins
JF - Proceedings of the National Academy of Sciences
JO - Proc Natl Acad Sci USA
SP - 12794
LP - 12799
DO - 10.1073/pnas.1506449112
VL - 112
IS - 41
AU - Gautam, Avneesh Kumar
AU - Panse, Yogesh
AU - Ghosh, Payel
AU - Reza, Malik Johid
AU - Mullick, Jayati
AU - Sahu, Arvind
Y1 - 2015/10/13
UR - http://www.pnas.org/content/112/41/12794.abstract
N2 - The complement system discriminates self from nonself targets solely owing to the presence of complement regulators on the host cells, which primarily belong to the regulators of complement activation (RCA) protein family. These regulators control complement activation on the host cell surface by two mechanisms termed “cofactor activity” (CFA) and “decay-accelerating activity.” Here, we have identified the critical structural determinants of an RCA protein responsible for imparting the CFA and show that these determinants bridge MG2 (macroglobulin-2) and CUB (complement C1r-C1s, Uegf, Bmp1) domains of C3b and interact with factor I. As a proof of principle, we show that incorporation of putative membrane cofactor protein (CD46) regions responsible for the bridging and factor I interaction in decay acceleration factor (CD55) results in gain of function. We, thus, define the molecular events that govern CFA.The complement system has evolved to annul pathogens, but its improper regulation is linked with diseases. Efficient regulation of the system is primarily provided by a family of proteins termed regulators of complement activation (RCA). The knowledge of precise structural determinants of RCA proteins critical for imparting the regulatory activities and the molecular events underlying the regulatory processes, nonetheless, is still limited. Here, we have dissected the structural requirements of RCA proteins that are crucial for one of their two regulatory activities, the cofactor activity (CFA), by using the Kaposi’s sarcoma-associated herpesvirus RCA homolog Kaposica as a model protein. We have scanned the entire Kaposica molecule by sequential mutagenesis using swapping and site-directed mutagenesis, which identified residues critical for its interaction with C3b and factor I. Mapping of these residues onto the modeled structure of C3b–Kaposica–factor I complex supported the mutagenesis data. Furthermore, the model suggested that the C3b-interacting residues bridge the CUB (complement C1r-C1s, Uegf, Bmp1) and MG2 (macroglobulin-2) domains of C3b. Thus, it seems that stabilization of the CUB domain with respect to the core of the C3b molecule is central for its CFA. Identification of CFA-critical regions in Kaposica guided experiments in which the equivalent regions of membrane cofactor protein were swapped into decay-accelerating factor. This strategy allowed CFA to be introduced into decay-accelerating factor, suggesting that viral and human regulators use a common mechanism for CFA.
ER -